285 In vitro cultivation of Anisakis simplex: pepsin increases survival and moulting from fourth larval to adult stage
L.IGLESIAS, A.VALERO, R.BENI; TEZ and F.J.ADROHER* Department of Parasitology, Faculty of Pharmacy, University of Granada, 18071-Granada, Spain
(Received 28 October 2000; revised 23 March 2001; accepted 23 March 2001) This paper describes the in vitro cultivation of the 3rd-larval stage (L3) of Anisakis simplex to adulthood in a much simpler and easier to prepare medium than those described to date. The adult males obtained are between 3n8 and 6n5 cm long and the females between 4n5 and 8n0 cm. Some individually cultivated females laid eggs which had an average size of 44n4i50n5 µm. The culture conditions were as follows: medium RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum and 1% commercial pepsin, at pH 4n0 and a temperature of 37 mC, and in air atmosphere with 5% CO#. The pepsin was found to be the key to the success of the culture. The average survival of the worms in the culture increased from 50 to 88 days, due to the fact that the survival of the adults practically doubled (increasing by 1n9 times). Furthermore, the number of worms that completed the 4th moulting (M4) increased by 4n2 times, from 22n9to95n6%. This culture medium may facilitate, due to its simplicity, the study of anisakids, or at least of A. simplex, constituting another step towards achieving a complete in vitro life-cycle for these parasites.
Key words: Anisakiosis, Anisakis simplex, in vitro cultivation, pepsin, moulting, survival.
(blue whiting), family Gadidae, purchased from the fish market of Granada (Southern Spain). The blue Anisakis simplex is a parasitic nematode of marine whiting, on the Atlantic and Mediterranean Spanish mammals, which infects a large variety of fish, coasts, is frequently parasitized by A. simplex s.l. among other hosts, during its larval stage. These (Ruiz-Valero et al. 1992; Valero et al. 2000). The parasitized fish may be ingested by humans, thus worms, found free in the host body cavity, were releasing the parasite larvae into the stomach, 20 mm or more in length, and were collected with possibly resulting in anisakiosis if the fish were not the help of a needle with a blunt tip, placed on a Petri adequately cooked. dish with 0n9% NaCl solution and washed in it In vitro cultivation of anisakids has had some several times. The worms were observed individually success, but is difficult to carry out due to the fact under an inverted microscope and those which that the culture media used to date are complex as showed any kind of internal or external damage were regards both their composition and their preparation discarded. They were then identified according to (Townsley et al. 1963; Van Banning, 1971; Grabda, morphological features (Hartwich, 1974; Petter & 1976; Carvajal et al. 1981; Likely & Burt, 1989, Maillard, 1988). Over 800 L3 were used in this 1992), study. Forty L3 (ca. 0n5% of total) of A. simplex s.l. Our aim was to develop a culture medium that was from the same hosts wee preserved at k70 mC for more defined in its composition and simpler to electrophoretic identification purposes. These larvae prepare, to make it easier to obtain adult parasites. In were analysed individually by isoenzyme electro- this paper, the defined medium used, supplemented phoresis using thick starch gel in a continuous buffer with fetal bovine serum and pepsin, allowed in vitro system (Nascetti et al. 1986; Martı!n-Sa! nchez, development of the 3rd larval stage (L3) of Anisakis Paniagua & Valero, 1998; Iglesias, Martı!n-Sa! nchez, simplex s.l. Rudolphi, 1809 to mature adults. Adroher & Valero, unpublished observations). Prior to cultivation, each larva was individually placed in an antibiotic-antifungal solution and axenized as described elsewhere (Iglesias, Valero & Adroher, 1997). Worms were cultured on a sterile The worms selected for our study were 3rd-stage polystyrene 24-well tissue-culture plate. The culture larvae of Anisakis simplex s.l. Rudolphi, 1809 isolated medium (1 ml) was placed into each well with 1 from the host Micromesistius poutassou Risso, 1826 parasite. The culture plates were then placed in an incubator at 37 mC and 5% CO# in humid air (except * Corresponding author: Departamento de Parasitologı!a, when indicated), and the culture medium was Facultad de Farmacia, Universidad de Granada, E-18071 Granada, Spain. Fax: j34 958 243862. E-mail: renewed twice a week. The worms were observed fadroher!ugr.es daily for mobility, moulting and survival.
Parasitology (2001), 123, 285–291. " 2001 Cambridge University Press DOI: 10.1017\S0031182001008423 Printed in the United Kingdom L. Iglesias and others 286
The culture medium (RPMI) was RPMI-1640 experiment in which the larvae were subjected to a plus 20% (v\v) heat-inactivated fetal bovine serum change in pH on the 5th day (from pH 7n2topH2n0, (IFBS). To the medium, where indicated, IFBS 3n0 and 4n0), when all the parasites had moulted to (40% v\v), horse blood (1% v\v), pepsin (1% p\v), L4. Moulting to L5 only occurred between days 43 -cysteine (4 m) or glutathione (4 m) were added and 71 in the medium in which the pH was changed and the worms cultivated at several pHs. to 4n0, 5 L5 were produced (20% of the initial larvae; The terms ‘maximum survival’ (Smax), ‘survival 3K,2L) with a SL& of 39n2 days. No statistically 50’ (S&!), and ‘average survival’ (Sav) in culture have significant differences were observed with the control previously been defined by us (Iglesias et al. 1997) as at pH 4n0 from the beginning of the culture (4 L5, follows: Smax is the day the last living nematode in 19% of the initial larvae; 2K and 2L; M4 between the experiment dies. S&! is the day on which 50% of days 54 and 64 of the culture; SL& 31n0 days). Other nematodes in the experiment are dead and Sav is the manipulations of pH produced suboptimal devel- arithmetic mean day of death of each nematode in opment. The high survival rates in these media at the experiment. The data are expressed as the pH 4n0 should be noted. meanpstandard error (..). Pepsin is a component of the gastric juice in the Similarly, other terms are defined as follows: ‘L5- stomachs of mammals. For this reason, we added survival’ (SL&) is the arithmetic mean of the days that pepsin to the culture media at various times during worms survive after the 4th to 5th stage moult (M4). cultivation. In the first assay, at pH 7n2, moulting to ‘L4-survival’ (SL%) is the arithmetic mean of the L5 did not occur with or without pepsin. However, days from M3 to M4. M4av is the arithmetic mean of when the assay was carried out at pH 4n0, there was the culture day on which the M4 of each nematode in a highly significant difference between the medium the experiment is completed. The data are expressed with or without pepsin added from the beginning −' as the meanp.. L4 indicates the larvae that (Sav, P ! 1i10 ; SL&, P ! 0n003). Some moulting completed M3 (i.e. the 3rd to 4th stage moult), and to L5 occurred in all media at pH 4n0 and reached L5 the larvae that completed M4 to adulthood. A 100% in the medium where pepsin was added at the statistical comparison of the culture data was made beginning. This M4 occurred between 20 and 60 using the Student’s t distribution. days of cultivation. The later the pepsin was added The media, sera and reagents were purchased to the culture, the lower the Sav and percentage from Sigma Chemical Co. (St Louis, MO, USA) and moulting to L5 (Table 2). Opposite results occurred Bio-Whittaker (Walkersville, MD, USA). Pepsin when the pepsin was eliminated from (i.e. not added was obtained from Probus (Barcelona, Spain). The to) the culture medium at different culture times commercial pepsin used in our experiments was (Fig. 2). On the other hand, 1 female, grown in the analysed by SDS–PAGE (Laemmli, 1970). Protein medium with pepsin added on the 14th day, began concentration was determined according to the ovoposition on the 97th day of cultivation, 51 days method of Lowry et al. (1951). after moulting to L5. This worm laid 15442 eggs in 16 days (with a maximum of 5672 eggs laid on 1 day). Other changes in the culture medium resulted in Forty L3 of A. simplex s.l. collected from M. reduced moulting and survival times compared with poutassou were electrophoretically identified as A. the pepsin-added media. The addition of either simplex s.s. (39) or A. pegreffii (1) (Nascetti et al. glutathione or -cysteine alone, to the culture 1986). medium inhibited the moult to L5. Even with The greatest moulting percentages to L4 were pepsin, -cysteine prevented the moult to L5 and obtained in the presence of 5% CO#, no matter glutathione significantly reduced the numbers which medium was used. The best parameters both moulting. Similarly, increasing the percentage of for survival and moulting occurred in the nutritive IFBS or adding horse blood alone, inhibited the medium RPMI (Table 1). Consequently, the fol- moult to L5. With pepsin and increased IFBS, the lowing experiments were carried out in the RPMI numbers moulting were reduced. In this experiment, medium in air atmosphere j5% CO#. in the control medium RPMI with pepsin, at pH 4n0, When the culture was carried out at different pHs, 22 worms moulted to L5 (91n6%) between days 22 it was observed that at any pH used, all or almost all and 46, of which 10 were male and 12 female, with a the L3 reach L4, but L5 were only produced at pH SL& of 56n1 days. One of the latter laid eggs on day 4n0(1K,3L;28n6% of the initial larvae). In this 101 (55 days after M4). The total number of eggs medium, the moult to L4 occurred between days 3 counted was 190793, laid during the period ranging and 4; the moult to L5 occurred between days 30 and from days 101 to 143 (42 days). The maximum 42, and SL& was 26n7 days (Fig. 1). number of eggs counted in 1 day was 16790 on day Moulting to L4 under natural conditions involves 113. a change from the pH in fish tissues to that in the Finally, the mean length of the adults obtained stomach of the final host. Therefore, we set up an was 4n4p0n3 cm for males (range 3n8–6n5 cm) and 4n9 In vitro cultivation of A. simplex 287
Table 1. Influence of 5% CO# on M3 and survival of Anisakis simplex larvae incubated in different media
Medium† nL3‡ Atmosphere Savp.. S&! Smax nL4(%)§
SS 18 Air 10n5p0n510161(5n5%) ns SS 24 Air j5%CO# 10n0p0n4 10 15 24 (100%) PBS 21 Air 7n3p0n5 7 13 0 PBS 26 Air j5% CO# 18n9p1n7* 19 35 22 (84%) RPMI 25 Air 6n4p0n2 7 10 0 RPMI 22 Air j5% CO# 24n9p0n8* 22 37 22 (100%)
†SS, 0n9% NaCl solution; PBS, phosphate-buffered saline, pH 7n2; RPMI, RPMI-1640j20% IFBS, pH 7n2. ‡Initial number of parasites. §Percentage of L3 that reached L4 stage. nL3 and nL4, Number of larvae in 3rd and 4th stages. Survival rates (maximum survival Smax, survival 50 S&!, and average survival Sav) as defined in the Materials and Methods section. .., Standard error. −& *Statistical comparison with the same medium without\with CO# using Student’s t distribution: P ! 1i10 ; ns, not significant.
Fig. 1. Effect of pH on the in vitro moulting (A) and survival (B) of Anisakis simplex larvae cultured on RPMI-1640 plus 20% IFBS. (A) Percentage of worms that completed M3 and M4 throughout the culture time at different pHs. (B) Percentage of worms alive throughout the culture time at different pHs. p0n2 cm for females (range 4n5–8n0 cm). The mean caught off the Atlantic coast of Spain where this size of the eggs was 44n4p0n5i50n5p0n7 µm(n l parasite is predominant over A. pegreffii (Nascetti et 50). al. 1986). To determine the purity of the commercial pepsin The maximum development and survival of the used in our experiments, it was analysed by SDS– larvae of A. simplex was obtained, as expected, in the PAGE (Laemmli, 1970). A single band of approxi- nutritive medium in the presence of 5% CO# mately 35 kDa was revealed. This band matched the (Table 1; Sommerville & Davey 1976; Mercer et al. band of purified and crystallized pepsin from Sigma 1986; Iglesias et al. 1997). Although the pH of the Chemical Co. No protein impurities were revealed in gastric juices in mammals is close to 2, we obtained the lane of pepsin from Probus. Other experiments a maximum survival and development in the culture performed with 0n01% pepsin (EC 3.4.23.1) from at pH 4n0. However, the gastric conditions in Sigma Chemical Co. showed similar results to ones cetaceans are different to those of land mammals. with 1% pepsin from Probus (results not shown). Cetaceans, final hosts of A. simplex, present a multi- chambered stomach, generally with 4 chambers. The worms are generally, but not exclusively, located in the non-glandular stomach or forestomach. This is Of the forty L3 analysed by electrophoresis, 39 were probably because it is the chamber in which the identified as A. simplex s.s.; 1 was identified as A. larvae usually are released from the food, when pegreffii. We thus assumed that all (or nearly all) bacterial digestion begins and due to the peristaltic larvae used in the experiments of the in vitro movements of this chamber. The larvae are fixed cultivation were A. simplex s.s., since all hosts were intimately to the gastric mucous where M3 occurs, L. Iglesias and others 288
Table 2. Influence of pepsin on the in vitro cultivation of Anisakis simplex larvae at pH 4n0 (nL3 and nL5 are the number of worms in 3rd larval or adult stage, respectively. Survival and moulting rates (average survival Sav, maximum survival Smax, survival 50 S&! and L5-survival SL5, average M4 M4av) as defined in the Materials and Methods section. .., Standard error).
M4avp.. Medium† nL3‡ Savp.. S&! Smax nL5 (%)§ (range) K:L SL5p..
Pepsin added at start 13 91n3p4n1*** 97 111 13 (100%) 39n1p2n8ns (20–52) 5:8 51n8p5n3** Pepsin, added on 5th day 15 87n0p6n5*** 85 134 13 (86n6%) 41n9p2n5* (27–61) 6:7 49n9p7n0* Pepsin, added on 14th day 11 63n8p11n2ns 48 158 6 (54n5%)¶ 38n0p4n7ns (25–59) 2:4 48n6p16n7ns Pepsin, added on 21st day 12 60n5p11n2ns 41 154 5 (41n6%) 31n8p2n9ns (25–38) 3:2 56n2p17n4ns Without pepsin 15 50n0p3n7 50 85 3 (20n0%) 34n3p0n3 (34–35) 2:1 31n5p2n4
†RPMI-1640j20% IFBS adjusted at pH 4n0. Pepsin is added at 1%. ‡Initial number of parasites. All worms reached L4 stage. §Percentage of L3 that reached L5 stage. ¶In this medium, 1 female laid 15442 eggs over 16 days. Statistical comparison with medium without pepsin using Student’s t distribution: *P ! 0n02; **P ! 0n003; ***P ! & 5i10− ; ns, not significant.
Fig. 2. Effect of pepsin (at 1% p\v) into the RPMI-1640 plus 20% IFBS medium on the in vitro development of Anisakis simplex larvae at pH 4n0. but the subsequent moult takes place in the stomach added, such as -cysteine and glutathione, the former lumen and, therefore, the adults are normally free or having been cited as improving the yield of the in only superficially attached to the stomach wall vitro cultivation of another anisakid, Contracaecum (Kikuchi et al. 1967 (cited by Podolska, Piusin! ski & osculatum, which matures in the stomach of seals Rokicki, 1997); Young & Lowe, 1969; Smith & (Likely & Burt, 1992). It might be worth noting that Wootten, 1978; Smith, 1989; Olsen, Aagnes & A. simplex is found in seal stomachs but does not Mathiesen, 1994a). However, Højgaard (1999) has reach maturity there whereas C. osculatum does recently reported a cluster of adults in the fore- (Young & Lowe, 1969; Smith & Wootten, 1978). stomach wall of a long-finned pilot whale. The pH This could also be related to the fact that the pH of of the forestomach ranges between 5n4 and 7n3 de- the monogastric stomach of seals during digestion pending on the diet of the cetaceans, at least in minke remains within the range of 1n5–3n5 (Mikkelsen, whales (Olsen et al. 1994a; Olsen et al. 2000). In the 1998), being lower than that recorded in the stomach second stomach or fundic chamber, where HCl and content of the whales. pepsin are produced, the pH is 5n0–5n3, whereas in In addition to the pH, the pepsin may also be a the other chambers the pH is 3n2–3n6 (Olsen et al. stimulus necessary in the development of anisakids, 1994b). at least of A. simplex, in the stomach of marine The change in pH, after M3 in vitro, did not lead mammals. On the one hand, L3 of A. simplex prove to any improvement in the results. Nor did the more resistant to digestion by pepsin than by other results improve when reducing substances were proteases of animal and vegetal origin in vitro In vitro cultivation of A. simplex 289
Table 3. Summary of the data on the in vitro cultivation of Anisakis simplex in RPMI-1640 (j20% IFBS) medium at pH 4n0 added with 1% pepsin (Results are expressed as meanpstandard error of the number ‘‘n’’ of experiments. For all these experiments 137 worms were used. Survival rates (average survival Sav, L4-survival SL4, and L5-survival SL5) as defined in the Materials and Methods section. The survival curves were significantly different by the Kaplan-Meier test (confidence 95%).%L5: Percentage of L3 that reached L5 stage.)
Medium (n) Sav SL4 SL5 %L5 K:L ratio
W\o pepsin (4) 50n3p0n438n2p5n230n2p1n222n9p2n21n02p0n35 With pepsin (3) 87n8p2n132n0p2n056n8p3n195n6p2n40n89p0n17 Student t distribution P ! 0n003 .. P ! 0n0003 P ! 0n00006 ..
.. Not significant
Table 4. Anisakis simplex in vitro culture in the literature
M3 M4 Egg size Adult length Reference Medium Conditions (%L4) (%L5) (µm) (cm)
Van Banning Pepsin digested 34–37 mC 4 or more days 26–98 days 40i50 K 3n5–7n0 (1971) beef liverj pH 2n0 p.i.† post-M3 K 4n5–15n0 beef blood Air (k) (k) Grabda (1976) Pepsin digested 36–37 mC 4–7 days p.i. 12–14 days 39–42i41–43 K 5n7–7n2 bovine liverj pH 2n0 (k) post-M3 L 5n6–10n4 fresh cattle blood* Air (50%) Sommerville & Medium 199 37n5 mC 3–5 days p.i. No No No Davey (1976) pH 7n0 (96n7%) Airj5% CO# Carvajal et al. Homogenated 34 mC Yes Yes –– (1981) fresh bovine liver* pH 2n0 (k) (42%) Air Iglesias et al. RPMI-1640j 37 mC 4–5 days p.i. No No No (1997) fetal bovine pH 7n2 (100%) serum Air j5% CO# This work RPMI-1640j 37 mC 3–4 days p.i. 16–57 days 44n4i50n5 K 3n8–6n5 fetal bovine pH 4n0 (100%) post-M3 L 4n5–8n0 serumjpepsin Airj5% CO# (95n6%)
*These media were based on the medium of Van Banning (1971). †p.i., Post-inoculum. –, Not reported.%L4: Percentage of L3 that reached L4 stage.%L5: Percentage of L3 that reached L5 stage.
(Dziekon! ska-Rynko, Rokicki, & Jabłonowski, 1997). although it does not appear to be necessary in the On the other hand, although the secretion of various first days of in vitro cultivation of L3. According substances with proteolytic activity have been de- to these results, it seems that the pepsin is required, scribed in anisakids (Sakanari, 1990), to our knowl- if not by L3, then by L4 from when this larva edge none similar to pepsin has yet been cited, which is formed and possibly by adults, as these have a could obviate the need for an exogenous source of greater mean survival rate SL& in media with pepsin this enzyme. (48–56 days) than in the control without it (31 days). Furthermore, the majority of the media used for It has also been demonstrated that the proteases play the cultivation of A. simplex and other gastric an important role in the ecdysis process of some nematodes are complex media which at some point nematodes, as they are involved in the digestion in their elaboration require a pepsic digestion of one processes of the old cuticle, enabling it to be expelled of their components (Van Banning, 1971; Grabda, (Rogers, 1970; Matthews, 1982; Lustigman et al. 1976). In no case is there any suggestion of the total 1996; Rhoads, Fetterer & Urban, 1997). elimination of the pepsin. Indeed, pepsin has been In summary, CO#, pH and pepsin are factors used in the in vitro cultivation of other gastric which influence the development of A. simplex,at nematodes (Douvres & Malakatis, 1977; Douvres, least in vitro. The results obtained by us using this 1979). medium, which is simpler than that used up to now Additionally, it can be affirmed that the addition of by other authors, show it to be useful for cultivation, pepsin favours the development of the parasites the as the yield, measured as a percentage of adults earlier it is added to the maintenance medium, obtained, is much higher than that obtained by other L. Iglesias and others 290 authors, when reported, and the size of the adults is , . . , . . . (1989). Cultivation of similar, although the maximum length of the females Pseudoterranova decipiens (sealworm) from third-stage obtained by other authors is greater than that larvae to egg-laying adults in vitro. Canadian Journal obtained in our cultures (Tables 3 and 4). of Fisheries and Aquatic Sciences 46, 1095–1096. Further studies are necessary to clarify the effect , . . , . . . (1992). In vitro cultivation of pepsin and other factors on these parasites and to of Contracaecum osculatum (Nematoda: Anisakidae) Canadian achieve an in vitro complete life-cycle of the anisakids from third-stage larvae to egg-laying adults. Journal of Fisheries and Aquatic Sciences 49, 347–348. in the laboratory. , . ., , . ., , . , . . (1951). Protein measurement with the Folin Thanks are due to Dr V. Dı!az-Sa! ez for advice with phenol reagent. Journal of Biological Chemistry 193, SDS–PAGE and Dr J. Martı!n-Sa! nchez for assistance with 265–275. the isoenzyme analysis and the Kaplan-Meier statistical test. The authors are also grateful to Drs M. A. Olsen , ., c, . ., , ., , ., , (University of Tromsø, Norway) and D. P. Hojgaard ., , . , . (1996). Cloning of a (Soelfjoerdur, Faroe Islands) for their help in compiling cysteine protease for the molting of Onchocerca the scarce Soelfjoerdur, existing data on the physiological volvulus third stage larvae. Journal of Biological conditions of the stomachs of cetaceans and pinnipeds. Chemistry 271, 30181–30189. This work was partly funded by the Spanish PB98-1312 !-! , ., , . , . (1998). from the DGESIC and the Research Groups Grant from Contribution to the knowledge of Hysterothylacium Junta de Andalucı!a. We are grateful to Mrs Gillian C. aduncum through electrophoresis of the glucose Butcher for the translation into English. phosphate isomerase and phosphoglucomutase. Parasitology Research 84, 160–163. , . . (1982). Behaviour and enzyme release by Anisakis sp. larvae (Nematoda: Ascaridida). , ., , ., , . , . Journal of Helminthology 56, 177–183. (1981). In vitro culture of larval anisakid parasites of , . ., , . ., , . , . . the Chilean hake Merluccius gayi. Journal of (1986). Cuticle production and ecdysis in larval Parasitology 67, 958–959. marine ascaridoid nematodes in vitro. Parasitology 92, , . . (1979). In vitro development of 711–720. Trichostrongylus axei, from infective larvae to young , . (1998). Summer diet of grey seals adults. Journal of Parasitology 65, 79–84. Halichoerus grypus in the Faroe Islands. Thesis. , . . , . . (1977). In vitro Department of Aquatic Resources and Environmental cultivation of Ostertagia ostertagi, the medium Biology. Norwegian College of Fishery Science, stomach worm of cattle. I. Development from University of Tromsø, Norway. infective larvae to egg-laying adults. Journal of , ., , ., , ., , . ., Parasitology 63, 520–527. , . , . (1986). Electrophoretic ! -, ., , . , . studies on the Anisakis simplex complex (Ascaridida: (1997). The impact of animal and plant proteases on Anisakidae) from the Mediterranean and North-East Nematoda 3rd stage Anisakis simplex larvae. Atlantic. International Journal for Parasitology 16, Oceanological Studies 1, 103–107. 633–640. , . (1976). Studies on the life cycle and , . ., , . . , . . (1994a). morphogenesis of Anisakis simplex (Rudolphi, 1809) (Nematoda: Anisakidae) cultured in vitro. Acta Digestion of herring by indigenous bacteria in the Ichthyologica et Piscatoria 6, 119–139. minke whale forestomach. Applied and Environmental , . (1974). Keys to genera of the Microbiology 60, 4445–4455. Ascaridoidea, no. 2. In CIH Keys to the Nematode , . ., , . ., , . . , . . Parasites of Vertebrates (ed. Anderson, R. C., (1994b). Functional anatomy of the gastrointestinal Chabaud, A. G. & Willmott, S.), pp. 1–15. CAB, system of Northeastern Atlantic minke whales Slough. (Balaenoptera acutorostrata). Journal of Zoology 234, , . . (1999). No significant development of 55–74. Anisakis simplex (Nematoda, Anisakidae) eggs in the , . ., , . ., , . . ., , . intestine of long-finned pilot whales, Globicephala , . . (2000). Chitinolytic bacteria in the melas (Traill, 1809). Sarsia 84, 479–482. minke whale forestomach. Canadian Journal of , ., , . , . . (1997). Some Microbiology 46, 85–94. factors which influence the in vitro maintenance of , . . , . (1988). Larves d’ascarides Anisakis simplex (Nematoda). Folia Parasitologica 44, parasites de poissons en Me! diterrane! e occidentale. 297–301. Bulletin du Museum National de Histoire Natural de , ., , ., , ., , . Paris 4se! r., 10[A], 347–369. , . (1967). Experimental studies of the , ., ! , . , . (1997). immunopathology of the intestinal anisakiasis. Igakuno Stomach wall changes caused by the presence of Ayumi 62, 731–736 (in Japanese). third-stage Anisakis simplex B larvae in infected dogs. , . . (1970). Cleavage of structural proteins Acta Protozoologica 42, 241–247. during the assembly of the head of bacteriophage T%. , . ., , . . , . . r. (1997). Nature, London 227, 680–685. Secretion of an aminopeptidase during transition of In vitro cultivation of A. simplex 291
third- to fourth-stage larvae of Ascaris suum. Journal larva of Anisakis sp. (Nematoda: Ascaridoidea). of Parasitology 83, 780–784. International Journal for Parasitology 6, 433–439. , . . (1970). The function of leucine , . ., , . ., , . . , aminopeptidase in exsheathing fluid. Journal of . . (1963). The in vitro maturation of the parasitic Parasitology 56, 138–143. nematode Terranova decipiens, from cod muscle. -, ., , ., , . . , . . Journal of Fisheries Research of the Board of Canada (1992). Presencia de asca! ridos en peces comerciales de 20, 743–747. frecuente consumo en Granada. In In Memorian al , ., !-! , ., -, . Profesor Doctor D F de P Martınez Gomez . . . T T (ed. , . . (2000). Larval anisakids parasitizing the Hernandez, S.), pp. 335–349. Cordoba, Spain, ! ! blue whiting (Micromesistius poutassou) captured from Universidad de Co! rdoba. Motril bay in the Mediterranean region of southern , . . (1990). Anisakis – from the platter to the Spain. Journal of Helminthology 74, 361–364. microfuge. Parasitology Today 6, 323–327. , . . (1989). Ulcers associated with larval , . (1971). Some notes on a successful Anisakis simplex B (Nematoda: Ascaridoidea) in the rearing of the herring-worm, Anisakis marina L. forestomach of harbour porpoises Phocoena phocoena (Nematoda: Heterocheilidae). Journal du Conseil (L.). Canadian Journal of Zoology 67, 2270–2276. International de l’Exploration de la Mer 34, 84–88. , . . , . (1978). Anisakis and , . . , . (1969). Larval nematodes from anisakiasis. Advances in Parasitology 16, 93–163. fish of the subfamily Anisakinae and gastro-intestinal , . . , . . (1976). Stimuli for lesions in mammals. Journal of Comparative Pathology cuticle formation and ecdysis in vitro of the infective 79, 301–313.